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Characterisation of five GH16 glycanase and transglycanase activities and of their hemicellulosic substratesSimmons, Thomas J. January 2014 (has links)
Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.
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Transglucosylation of cell wall polysaccharides in equisetum fluviatileMohler, Kyle Edward January 2012 (has links)
Plant cell walls determine cellular shape and provide structural support for the entire plant. Polysaccharides, comprising the major components of the wall, are actively remodelled throughout development. Xyloglucan endotransglucosylase (XET)/hydrolase (XTH, EC 2.4.1.207) cleaves xyloglucan (XyG), the donor substrate, and attaches a portion to another XyG chain, the acceptor substrate. Recently, a novel transglucosylase called mixed-linkage β-glucan (MLG) : XyG endotransglucosylase (MXE) was discovered in horsetails (Equisetum spp.) that could attach a portion of MLG to XyG, resulting in a hetero-polymer product. My aims were to further investigate the nature of this activity, biochemically characterize the enzyme, and explore its physiological role. MXE activity was attributable to an enzyme unlike Equisetum XTHs. MXE had a p1 of 4.1 (XTHs were 6.6-9), a pH optimum of 6.3 (XTHs preferred 5.5), and had higher activity using smaller oligosaccharide acceptor substrates like XXXGol (XTHs were more active using XLLGol). Importantly, the MXE protein was shown to utilize both MLG and XyG as donor substrates, and therefore have both MXE and XET activity. Also, the enzyme was capable of using various glucan oligosaccharides (O) as substrates, including MLGO, XyGO, and cello-O, but not laminari-O. By using a novel ex vivo approach, the proportion of extractable MXE product to XET product was found to increase in older tissues. Transglucosylase products were localized in sclerenchyma and structural parenchyma by in situ assays, implying a strenghening function for MXE. Surprisingly, another novel activity was discovered that could covalently attach cellulose to XyG, and termed cellulose : xyloglucan endotransglucosylase (CXE). This activity was attributed to the MXE enzyme, implying that the protein is a promiscuous endotransglucosylase. The presence of CXE in other plants has not yet been tested. Besides being a novel discovery in plant cell biology, the modification of cellulose has applications in a number of industries.
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An Investigation of ßglux, a Glucosidase Co-Expressed with Cslf6 in Oat (Avena sativa) and Barley (Hordeum vulgare)Gines, Michael Christopher 01 December 2016 (has links)
Mixed Linkage Glucan (MLG, or (1,3;1,4)-ß-D glucan) is a component of cell walls for major cereal crops and is significant to food and beverage industries. To better understand genetic factors affecting MLG content in oats, this study investigates the presence of glucosidases likely to participate in MLG production. A glucosidase showing co-expression with CslF6—the primary gene responsible for MLG synthesis—could indicate a hand in MLG production by association. Reference genes for expression analysis as well as glucosidase candidates were first selected using in silico methods. In both cases, barley was used as model species because it has abundant public bioinformatic resources for in silico data mining, and it generates large amounts of MLG, like oats. Actin, malate dehydrogenase, and elongation factor 2, were validated in oat and barley as top reference genes. They were then used to compare the expression activity of the top glucosidase candidate gene, ßglux, with CslF6. ßglux was found to have increased activity with CslF6 during caryopsis development. It is a strong candidate for future transgenic experiments regarding its effect on MLG production.
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